In Chapter 4, we describe in detail the remarkable experiments that established once and for all that DNA is the genetic material. This recognition, together with the Watson–Crick structure of DNA, meant that by the mid-1950s a molecular theory of genetics had begun to develop. Much of the early work in this direction depended on the use of bacteria and viruses, particularly those viruses that infect bacteria, the bacteriophages or phage in scientific jargon.
The genetics of phage and bacteria had long been neglected, for neither follows the laws of classical Mendelian genetics. Both are haploid under most circumstances. However, in 1943, microbiologist Salvador Luria and physicist Max Delbrück provided convincing evidence of mutations in bacteria. In the same year, a major technical breakthrough was accomplished by Joshua Lederberg and Edward Tatum in the discovery of bacterial conjugation (Figure 1). In this process, one bacterium inserts all or part of its DNA into another, followed by recombination of the two DNA molecules. With some strains, called high frequency of recombination strains, practically all of the donors are active, and conjugation can be synchronized. If conjugation is halted at a series of different times, different amounts of DNA will have been transferred, permitting recombination of only those genes that have been transferred. This provided a convenient way to map genes on the bacterial chromosome before powerful sequencing methods were available.
Fig1. Bacterial conjugation. Conjugation between two bacterial cells can occur only when one of the partners carries the F or fertility plasmid; these cells are known as F-positive or F+ cells. The F plasmid exists as an episome, that is, independently of the main bacterial chromosome. It carries its own origin of replication, an origin of transfer where nicking occurs to initiate transfer to a recipient F– cell, and a whole battery of genes responsible for formation of the pilus and attachment to the recipient cell. (A) Steps in the process of conjugation are as follows: (Step 1) Pilus attaches to recipient cell and brings the two cells together. Most probably, the pilus is not directly used as a transfer channel. The channel is formed through the action of a specific enzyme at the base of the pilus, which initiates membrane fusion. (Step 2) The F plasmid is nicked and the nicked strand is unwound from the intact strand; transfer to the recipient cell begins. (Step 3) Single-stranded DNA is transferred to the recipient and copied to produce a double-stranded F plasmid; the single-stranded F plasmid in the donor cell is simultaneously copied to produce a double-stranded F plasmid. Sometimes, the F plasmid is integrated into the genome of the donor; these strains are known as high frequency of recombination or Hfr strains. In such cases, the entire bacterial chromosome or a part of it can be transferred into the recipient cell. The amount of chromosomal DNA transferred depends on how long the two conjugating bacteria stay in contact: transfer of the entire chromo some normally requires ~100 minutes. Homologous recombination allows for integration of the transferred chromosome into the genome of the recipient cell. (B) Electron micro graph of two bacterial cells in the process of conjugation. [B, courtesy of Charles Brinton and Judith Carnahan, University of Pittsburgh, PA, USA.]
